Research Reports No.25 （2019）

25－01

Applied Research on Antioxidant Action of Existing Additive Chitosan

Katsuhiko Yoshizawa, Akiko Takenouchi
Department of Food Sciences and Nutrition, School of Human Environmental Sciences,Mukogawa Women's University

Acute hepatitis showed the widespread necrosis with the progressive failures such as jaundice, bleeding tendency and hepatic encephalopathy. It is important to delay the progression of the clinical condition by the immediate onset of treatment. In this research, the effects on antioxidant action of the existing additive chitosan was tested in two different types of acute liver injury models, as follows: a green tea extract-induced and a carbon tetrachloride-induced models in rats. In these models, the oxidative stress was proved to be involved in the pathogenesis of the liver pathology, similarly to what is seen in human acute hepatitis. The orally absorbable chitosan oligosaccharide was administered. The results of the investigation indicate that the chitosan oligosaccharide reduced the severity of liver injury in these models by the antioxidant activity.

25－02

Chemical synthesis of existing food additives for internal reference Standards

Yosuke Demizu
Division of Organic Chemistry, National Institute of Health Sciences

For quality assurance of food additives, it is important to establish compositional standard tests by developing highly accurate analytical and evaluation methods. In this research, a new analytical method is established by developing a total synthetic route of some food additives for qualitative or quantitative standards,which are difficult to set content standards using conventional methods. The purpose of this research is developing the simple and accurate standard testing methodology. In this paper, the synthetic methods for lycopene, crocin, and curcumin are described.

25－03

Risk assessment of arsenic in food: analysis of mechanisms of arsenic carcinogenicity

Min Gi
Osaka city university graduate school of medicine, Department of molecular pathology

In our previous studies, we found that lung tumors were increased in the male mice exposed to organic arsenic compound dimethylarsinic acid (DMA) at the fetal stage. We also found a significant increase in histone H3K9me3 in the lungs of male newborn mice exposed to DMA transplacental exposure. However,its target genes remain to be clarified. In this study, we determined the target genes of histone H3K9me3 in the lung of male neonatal mouse exposed to DMA transplacental by microarray analysis. DMA was administered to pregnant female CD-1 mice at a dose of 0, 200 ppm from gestation day 8 to 18, and lungs were collected from the obtained male neonates and 6-week old mice. Analysis of neonatal mouse lung revealed that X chromosome was one of the targeting region of H3K9me3. Microarray analysis showed that expression of Xist, a X chromosome-inactivated lncRNA, was increased in the DMA exposure group. Furthermore, pathway analysis indicated that Fibrosis and Glucose metabolism pathways were activated, and Lipid metabolism pathway was inactivated. In 6-week old mouse lung, Ki67 index was significantly increased in the DMA transplacental exposure group. Our results showed that epigenetic changes by DMA exposure play import roles in mouse lung carcinogenesis.

25－04

The Effect of flavonoid-containing additives on hepatitis C virus Production

Koichi Watashi
Department of Virology II, National Institute of Infectious Diseases

Hepatitis C virus (HCV) modifies host cellular function to maintain persistent infection and induce pathogenic condition, including the accumulation of hepatic lipid droplets, which serves for the efficient production of progeny viruses and the elevated risk for developing tumor. In this study, by using HCV production cell culture system, we identified flavonoids that reduced HCV production from hepatocytes. These flavonoids repressed the transcriptional activity of aryl hydrocarbon receptor (AhR) and blocked the induction of its downstream gene, cytochrome P450 1A1 (CYP1A1), which are reported to induce hepatic lipid droplets. We also identified the structures important for the anti-HCV and the anti-lipid production effect. These results suggest the potential usefulness of flavonoids to prevent and control HCV-related diseases.

25－05

Safety evaluation of food additives using a novel detection system of epigenetic mutagen

Kei-ichi Sugiyama
Division of Genetics and Mutagenesis, National Institute of Health Sciences

Carminic acid, an anthraquinone glucoside, is a major pigment in cochineal extract which has been used as a natural red dye for food. It has been reported that alizarin which is an anthraquinone derivative has a potency of dysfunction for epigenetic regulation. In the present study, epigenetic toxicity of carminic acid was investigated using a yeast-based bioassay that can detect epigenetic alterations contribute to promoter activity of a flocculation gene FLO1. The bioassay, the so called "FLO assay", revealed that carminic acid (5-20 μM) significantly enhanced the reporter activity under the transcriptional control of the FLO1 promoter in control strain and yeast transformed with human DNA methyltransferase cDNAs. The flocculation level of control strain was dosedependently increased by carminic acid (10-40 μM). These results suggest that carminic acid could exert epigenetic effects. However, mammalian cell studies to evaluate epigenetic toxicity of carminic acid are still required.

25－06

Risk reduction of type 2 diabetes by intake of a food additive, myristic acid

Fumio Sakane
Department of Chemistry, Chiba University Graduate School of Science

Decreased levels of the δ isozyme of diacylglycerol kinase (DGK) in skeletal muscle attenuate glucose uptake and,consequently, are critical for the pathogenesis of type 2 diabetes. We recently found that free myristic acid (14:0), but not free palmitic acid (16:0), increased the DGKδ protein levels and enhanced glucose uptake in C2C12 myotube cells. However, it has been unclear how myristic acid regulates the level of DGKδ2 protein. In the present study, we characterized the myristic acid-dependent increase of DGKδ protein. A cycloheximide chase assay demonstrated that myristic acid, but not palmitic acid, markedly stabilized DGKδ protein. Moreover, other DGK isozymes, DGKη and ζ, as well as glucose uptake-related proteins, such as protein kinase C (PKC) α, PKCζ, Akt and glycogen synthase kinase 3β, failed to be stabilized by myristic acid. Furthermore, DGKδ was not stabilized in cultured hepatocellular carcinoma cells, pancreas carcinoma cells or neuroblastoma cells, and only a moderate stabilizing effect was observed in embryonic kidney cells. A proteasome inhibitor and a lysosome inhibitor, MG132 and chloroquine,respectively, partly inhibited DGKδ degradation, suggesting that myristic acid prevents, at least in part, the degradation of DGKδ by the ubiquitin-proteasome system and the autophagy-lysosome pathway. Overall, these results strongly suggest that myristic acid attenuates DGKδ protein degradation in skeletal muscle cells and that this attenuation is fatty acid-, protein- and cell line-specific.

25－07

Development of an analytical method for determination of non-volatile amines in fish and fish products using Py-Tag as a new derivatization reagent

Hiroshi Akiyama
Division of Foods, National Institute of Health Sciences

We investigated the analytical method of seven nonvolatile amines (histamine, tyramine, cadaverine, putrescine,spermidine, spermine and phenylethylamine) in fish and fish products by liquid chromatographytandem mass spectrometry (LC-MS/MS). A new derivatization reagent, 2,4,6-triethyl-3,5-dimethyl pyrylium trifluoromethanesulfonate (Py-Tag), was used for detection of the nonvolatile amines. We finally established a simple sample preparation method to determine four of the seven nonvolatile amines, histamine, tyramine,cadaverine and phenylethylamine, in fish and fish products for LC-MS/MS analysis. The four nonvolatile amines were extracted from fish and fish products with trichloroacetic acid and the diluted extract was derivatized with Py-Tag, and was then subjected to LC-MS/MS analysis. Calibration curves for the four nonvolatile amines had excellent linearity (r ＞ 0.999). The limits of quantification in the calibrations curves were 0.2 ng/ mL for histamine, tyramine and phenylethylamine and 1 ng/mL for cadaverine. The matrix effects derived from the tested fish and fish products were negligible in the LC-MS/MS analysis. The impact of the sample matrices on the Py-Tag derivatization was also negligible. Overall, these results suggested the present method is reliable and suitable for rapid analysis in the four nonvolatile amines in fish and fish products.

25－08

Analyzing the effect of food additives using a new monkey intestinel　organoid culture

Ken Iwatsuki
Tokyo University of Agriculture, Department of Nutritional Science and Food Safety

Artificial sweetener such as saccharin, aspartame or sucralose, are often used as food additives in various foods or beverages. These sweeteners bind to sweet taste receptors in the oral cavity and evoke sweet taste sensation.
However, their effect on gut epithelium is not well understood. In this study, we aim to investigate the effect of artificial sweeteners by introducing monkey enteroids. First, we attempted to generate monkey enteroids using a piece of gut epithelium shared by Primate Research Institute of Kyoto University.
By searching the condition which immediately switch the stage of proliferation/differentiation, we found that removing the growth factor like Wnt factors would promote cell maturation. We are ready to examine the effect of sucralose stem cell activity using the organoid.

25－09

Action research for domestic production of safe and high quality saffron:application for agritherapy and contribution to local resident's health

Kayoko Takaura
The Museum of Osaka University

Saffron (the stigma of Crocus sativus L.) is a highly economical farm product which has been valued as medicine and spice from ancient times. Taketa city (Oita, Japan) is known as its major production area, where "Taketa-Saffron Cultivation Method" has been developed. It is characteristic cultivation method that saffron blooms indoors without soil and water, while it is cultivated and harvested outdoors in the major saffron producing foreign countries. Highquality saffron has been produced by this method in Taketa city, but the number of saffron producers is decreasing because of economy and aging. In this study, we investigated the history and clarify the benefits of this method in order to encourage the technique succession and domestic production of saffron. Our field study and interviews in Taketa city revealed that "Taketa-Saffron Cultivation Method" was encouraged and improved by experienced farmers in that area. We collected saffron samples from Taketa city and abroad, then revealed the high quality of saffron produced in Taketa city through the morphological investigation. Now we are trying to record the environmental conditions for producing high-quality saffron. By sharing these results, we are supporting the inheritance and practice of this unique method.

25－10

Profiling of Components from Toxic Mushroom for the Obtainment of Chemical Standards

Koichi Inoue
College of Pharmaceutical Sciences, Ritsumeikan University

In this study, we are profiling the components from toxic mushroom for the obtainment of chemical standards. These chemical components were separated and purified by high-speed countercurrent chromatographic (HSCCC) technique. The HSCCC is useful, efficient and low-priced method to utilize the twosolvent system. Our project produces the chemical back and data base of various toxic mushrooms in Japan. First step, the major toxic mushroom such as Omphalotus japonicus was applied by our approach. Illudin S from mushroom such as Omphalotus japonicus and illudens is a natural sesquiterpene analog with strong anti-tumour and antiviral activities. However, it is difficult to obtain sufficient amount of high pure illudin S from natural product by simple, efficient and low-cost purification techniques. There, we offer to apply the HSCCC for preparative purification of illudin S from mushroom extract. For two-solvent system, the optimal condition of hexane/ethyl acetate/methanol/water (1/5/1/5, V/V/V/V) was optimized to obtain the pure illudin S from crude extract. This purified component was evaluated by liquid chromatography (highpurity > 99%) and tandem mass spectrometry. The yield amounts of illudin S (1.3 mg/about 10 g Omphalotus japonicus) at one running are determined by liquid chromatographic calibration. It is concluded that by requiring natural material and cost-effectiveness, our method represents a significant improvement over the complicated techniques for the purification of illudin S from natural materials. This evidence showed that our approach would achieve the profiling of components from toxic mushroom for the obtainment of chemical standards.

25－11

Study on effects of fructooligosaccharide and wheat bran on tissue concentration of mercury at assuming methylmercury exposure from fish consumption

Masaaki Nagano1, Masatake Fujimura2
1 Hygienic Chemistry Section, Department of Basic Medical Sciences,National Institute for Minamata Disease
2 Director for Research General Coordination, National Institute for Minamata Disease

Methylmercury (MeHg) is a neurotoxicant that exists widely in the natural environment. Since environmental MeHg is accumulated in seafood through the food web, consumption of fish and shellfish is the major source of MeHg exposure for humans. Because of fears regarding the adverse effect of MeHg contained in seafood on the developing fetus brain, pregnant women have been warned against consuming fish and shellfish in many countries. We had previously demonstrated that daily intake of wheat bran (bran) or fructooligosaccharide (FOS) significantly reduces tissue Hg levels after single administration of MeHg in female mice. In the present study, we examined the effect of bran and FOS on tissue Hg concentration at continuous administration of MeHg in female mice. As a result,continuous intake of bran or FOS accelerated the excretion of Hg and subsequently decreased tissue Hg levels in mice. These results suggest that daily intake of bran or FOS may reduce tissue Hg levels after MeHg exposure in animals and human.

25－12

Survey on food additives used as the functional substances of Foods with Functional Claims

Sayaka Masada
Division of Pharmacognosy, Phytochemistry and Narcotics, National Institute of Health Sciences

Food additives are substances generally added to food to preserve flavor or enhance its taste, appearance, or other qualities. Some food additives are recently used as food substances in "foods with functional claims." In this study,we tried to illuminate the actual states of food additives used in foods with functional claims to clarify and discuss the points of their regulation. Following last year's investigations, 12 kinds of food additives were found to be registered as the functional substances at the safety level. The analytical methods with higher degree of precision were egistered for functional substances than those in the Japanese Standards of Food Additives. Moreover, weight variation test,disintegration test and dissolution test using four marketed products revealed that the most of foods with functional claims containing monoglucosyl hersperidin as functional substances keep the physical property equivalent to that of pharmaceutical drugs.

25－13

Studies on the effect of food additives on detection of genetically modified food--the 2nd report

Kosuke Nakamura
National Institute of Health Sciences, Division of Biochemistry

We examined the applicability of DNAs extracted and purified from dried-fruits commodities to real-time polymerase chain reaction (PCR) methods for detecting unauthorized genetically modified food ingredients. An endogenous gene having a single copy present in the haploid papaya genome was undetected from all commodities that were food-labelled to contain sodium sulfite salt as a food additive. Real-time PCR targeting at a high copynumbered endogenous gene, such as 18S rDNA, was detected reproducibly. Five undred nanogram linear plasmid incubated with sodium sulfite at final concentrations of ≦ 0.10 M did not show DNA degradation. Sodium sulfite addition to final concentration of 0.25 M into ground fresh papaya reduced the DNA yield up to 15% using ionexchange column DNA purification method (Genomic-tip), and the total DNA yield recovered when the sample was adjusted pH to 8. These results showed that the food processing of the dried-fruits with the sodium sulfite causes the low DNA-detectability using real-time PCR.

25－14

Development of asparaginase from lactic acid bacteria for suppression of acrylamide formation in processed food and verification of its usability as a food additive

Mamoru Wakayama
Department of Biotechnology, College of Life Sciences, Ritsumeikan University

Asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia, and is present in various organisms from microorganisms to mammals. While asparaginase has been used for the treatment of acute lymphoblastic leukemia, it is receiving a lot of attention as a potent mitigating agent for reducing the acrylamide contained in processed food. Little studies have been reported on asparaginase from lactic acid bacteria. Therefore,we aimed at obtaining semi-comprehensive information of potential asparaginase from lactic acid bacteria which are usable for mitigation of acrylamide generation in processed food. In this study, lactic acid bacteria of sixteen species in eight genera were investigated for their asparaginase production. Lactobacillus sakei, Streptococcus thermophiles,and L. plantarum showed a relatively high asparaginase activity among them. Genes of asparaginases from S.thermophiles and L. plantarum have been cloned and expressed in Escherichia coli. We purified and characterized the recombinant enzymes and compared their properties with the other enzymes. We also screened plasmid DNA from several lactic acid bacteria for constructing gene expression system of lactic acid bacteria as a host cell and prepared lactic acid bacteria- Escherichia coli shuttle vector.

25－15

Regulatory effects of sweeteners (glucose, sucralose) on fat taste perception and preference

Keiko Morimoto
Department of Health Sciences, Faculty of Human Life and Environment, Nara Women's University

In modern life, high-fat diets, which are risk factors for obesity or hyperlipidemia, are widespread. It is important for human health to clarify regulation system of fat taste preference. However, it is unclear whether dietary fat or sweeteners modulate fat taste perception and preference. We examined whether fat or sweeteners stimulations of lingual sensors affect fat preference in healthy young Japanese men and women. Female subjects participated in laboratory experiments three different phases during the menstrual cycle. There was no gender difference or fluctuation during menstrual cycle in oleic acid detection threshold determined by a three-alternative forced-choice methodology or fat preference assessed by choosing cottage cheese containing four different fat concentrations. The lingual stimulation with dietary fat or sucrose tended to increase, or increased the fat preference. In addition, estrogen replacement reduced fat preference and suppressed fat intake, but had no effects on lingual CD36 protein and mRNA expression in ovariectomized rats. The present study suggests that oral fat preference is increased after dietary fat or sweetener in young men, but not in young women.

25－16

Refinement of hepatocarcinogenicity evaluation for furan derivatives based on the biological characterization of rat preneoplastic lesions

Shinji Takasu
Division of Pathology, Biological Safety Research Center, National Institute of Health Sciences

Furan induces hepatocellular tumors in rodent. It has been reported that the mutant frequencies of reporter gene are not changed in the liver of furan-treated gpt delta rats. In contrast, the number and area of glutathione S-transferase placental form (GST-P)-positive foci are significantly increased. We previously reported that GST-P positive foci induced by diethylnitrosamine were increased after cessation of the treatment, those by furan being decreased. In present study, to establish the method to distinguish two different types of GST-P positive foci in terms of their kinetics after cessation of carcinogen treatment, we investigated the kinetics of GST-P positive foci induced by 2-methylfuran (2-MF) or 2-pentylfuran (2-PF). 6-week old male F344 rats were given 30 mg/kg/day 2-MF or 100 mg/kg/day 2-PF by gavage for 13 weeks. Quantitative analysis for GST-P positive foci was performed just after the cessation of the treatment and 7 weeks later. Number and area of GST-P positive foci in the liver of rat treated with 2-MF or 2-PF were decreased after cessation. These results indicated that 2-MF and 2-PF might be similar to furan in terms of formation of GST-P positive foci.

25－17

Study on preparation of reference materials for analysis of ciguatoxins,principal toxins of ciguatera fish poisoning

Naomasa Oshiro
Division of Biomedical Food Research, National Institute of Health Sciences

Availability of ciguatoxins, causative agents of ciguatera fish poisoning, are extremely limited. The flesh of fishes,Lutjanus bohar and Gymnothorax isingteena were used to prepare Reference Materials of Fish Powder containing Ciguatera Poison. These reference materials contain CTX1B, 52-epi-54-deoxyCTX1B and 54-deoxyCTX1B. Since these three congeners are principal toxins of ciguatera fish poisoning occurred in Okinawa and Amami, these materials will be useful for ciguatoxin analysis. These reference materials will be provided to the public health laboratories.

25－18

Study of the mechanism of antifungal activity of ε-poly-L-lysine, a food additive, which is characterized by overaccumulation of chitin

Yoshiharu Inoue
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University

The mode of antifungal activity of ε-poly-L-lysine (ε-PL), a food additive, was investigated using the budding yeast Saccharomyces cerevisiae as a model organism of fungi. S. cerevisiae mutants defective in the Mpk1-MAP kinase cascade showed an increased resistance against ε-PL. A mutant defective in cell wall stress sensors, which lie upstream of the Mpk1-MAP kinase cascade, did not exhibit the resistance to ε-PL. Synthesis of chitin, one of the cell wall components of yeast, was enhanced following the treatment with ε-PL, and the resistance to ε-PL was observed in a mutant defective in chitin synthase. Activation of Mpk1- MAP kinase cascade occurred following the treatment with ε-PL, which was also observed in the cell wall stress sensor mutant. Taken together, ε-PL activates the Mpk1-MAP kinase cascade through an unknown pathway that is different from the already-known pathway, and overaccumulation of chitin is likely to be one of the causes that exerts antifungal activity of ε-PL.

25－19

Chemical approach for the isolation of curcumin metabolizing bacteria

Toshio Niwa
Faculty of Health and Nutrition, Shubun University

We have isolated a metabolite of curcumin produced by human feces. From the analytical data, we determined the structure as 3-hydroxy-1,7-bis(3,4-dihydroxyphenyl)heptane (2). We herein synthesized the possible intermediates to determine the metabolic pathway and to isolate becteria that relate to curcumin metabolism. We synthesized tetrahydrocurcumin, hexahydrocurcumin, octahydrocurcumin, and demethyltetrahydrocurcumin. The bacteria named 1C produced 1,7-bis(3,4-dihydroxyphenyl)heptane from all synthetic curcumin derivatives. We then tried to isolate the bacteria which can produce hexahydrocurcumin from curcumin or which can metabolize demethyltetrahydrocurcumin. We then purified a colony that metabolizes demethyltetrahydrocurcumin.

25－20

Correlation between pectin polysaccharide structure and regulation mechanism of gastrointestinal function

Tomio Yabe
Faculty of Applied Biological Sciences, Gifu University

Pectin, a water-soluble dietary fiber, has a side chain consisting of a plurality of neutral sugars with galacturonic acid as the main chain. While intake of pectin passes through the small intestine, pectin that is not digested and absorbed directly acts on intestinal epithelial cells in a specific manner for its polysaccharide structure, changing the morphology of villi and increasing the area occupied by intestinal epithelial cells in villi. This indicates that the absorption efficiency of nutrients in the intestinal tract may be regulated by the action of non-nutrient pectin. In this study, mouse intestinal organoids were constructed as an experimental model to reproduce the physiological gastrointestinal tract, and sugar beet pectin characterized by a special polysaccharide structure was used to compare the effect on intestinal organoids with other pectin. In addition, the correlation between the polysaccharide structure and the function of pectin was examined by the modified pectin whose emulsifying property and emulsifying stability were improved by maturation technology. Citrus and sugar beet-derived pectins having different polysaccharide structures are likely to have opposite reactivities to the growth of intestinal organoids. On the other hand, in the pectin whose emulsifying stability was improved by moist heat treatment of sugar beet pectin, the characteristics of the natural pectin were diminished and the characteristics similar to citrus pectin were exhibited. In future, analyzing the mechanism at the molecular levels using intestinal organoids are required.

25－21

Inhibitory effects and mechanism of food additives derived from polyphenols on the bacterial superantigen toxin

Yuko Shimamura
School of Food and Nutritional Sciences, University of Shizuoka

Staphylococcal enterotoxin A (SEA) is a toxin protein, and is the most common cause of staphylococcal food poisoning. Polyphenols, such as catechins, are known to interact with proteins. In this study, the effects of the food additives (Teavigo; a green tea polyphenol preparation containing 94% (−)-epigallocatechin gallate (EGCG)) on SEA-inducible genes were analyzed using Real time RT-PCR and DNA microarrays. As a result, exposure of SEA to mouse spleen cells significantly induced a response of Th1 cells. In contrast, Theavigo down-regulated the expression levels of the inflammatory mediators (IL-12p40, ISG15, IRF3, IFN-γ and ZBP1) induced by SEA. These results suggest that Theavigo has a negative feedback regulatory action on the response of Th1 cells induced by SEA. Next,the effects of Teavigo on SEA-induced STAT3 phosphorylation was examined, Teavigo significantly suppressed STAT3 phosphorylation. Data from Western blot analysis suggest that the hydroxyl group at position 3 of the galloyl group in the EGCG structure was responsible for binding SEA and the suppresses the SEA-induced phosphorylation of STAT3. Moreover, the interaction between SEA and Teavigo was also observed under various pH (pH 4.0 to 8.0) conditions and in the coexistence of bovine serum albumin. In addition, the interaction between SEA interaction of EGCG and the gp130 receptor activating STAT3 was analyzed using protein thermal shift assay. The interaction between EGCG and SEA inhibited the binding of gp130 receptor to SEA and inhibited the phosphorylation of STAT3. Our results provide further understanding of the binding interactions between the food additives derived from polyphenols and SEA, and the inhibition of Bacterial superantigen toxin activities by the food additives derived from polyphenols.

25－22

Bioavailability of ellagitannins with antiglycation properties

Hideyuki Ito
Department of Nutritional Science, Faculty of Health and Welfare Science,Okayama Prefectural University

Ellagitannins having characteristic acyl units such as gallagyl and valoneoyl groups contain several food additives. The presence and the biological activities of the metabolites derived from these acyl units have been not reported. Furthermore, bioavailability of a main ellagitannin metabolite, urolithin A, was also little known. In the present study,we investigated bioavailability of urolithin A after oral administration to rats. After administration of urolithin A (1 or 10 mg) to rats, plasma, urine and feces were collected to 48 hours after administration. The collected samples were extracted with ethyl acetate and dried. The conjugated and non-conjugated urolithin A were quantified in the biofluid samples by using HPLC and HPLC-MS/MS methods. Urolithin A was mostly present as conjugate form in plasma,but conversely mostly free form in urine. In addition, plasma concentration of urolithin A reached maximum at 3 to 6 hours after administration, and urinary excretion was also excreted 48 hours. These findings were suggested that urolithin A circulated in the entero-hepatic circulation after absorption and was excreted in urine after being deconjugated in the kidney. In this study, we provided basic scientific data for bioavailability of functional ellagitannin metabolite.

25－23

Effects of sweeteners on the pathosis and underlying mechanisms of lifestylerelated diseases for the molecular target control of such diseases

Noriko Kemuriyama
Department of Nutritional Science and Food Safety, Faculty of Applied Biosciences,Tokyo University of Agriculture

The aim of the present study is to examine effects of sweeteners on the pathosis and underlying mechanisms of lifestyle-related diseases using animal models. In the present study, we chose non-alcoholic steatohepatitis (NASH) and inflammatory bowel disease (IBD) as diseases, Luo Han Guo fruit extract as a sweetener, and rat and mouse models featuring a chronic feeding of a choline-deficient, methioninelowered, l-amino acid-defined (CDAA) diet and murine dextran sulfate sodium colitis model as animal models. Luo Han Guo (Siraitia grosvenorii> fruit is wellknown for its sweet taste, and its extract is widely used as a sweetener and a component of health-care products, such as antitussive and anti-sore throat agents. In southern China, Luo Han Guo has been believed to aid longevity. We conducted 3 experiments in the fiscal year of 2018, and in experiment 1, we evaluated the effect of Luo-Han-Guo fruit extract on the NASH like lesion induced by feeding CDAA diet in Fischer 344 (F344) rats. As a result, no toxicological changes were observed in the basal diet group due to the administration of Luo-Han-Guo fruit extract at 2 weeks and 6 months. while the CDAA diet administration group showed hepatocellular injury from 2 weeks including elevated plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity,discoloration of the liver in gross pathology, fatty change of hepatocyte, slight inflammation and fibrosis, increased hepatocellular apoptosis in histopathologically. In addition, roughening of liver surface and marked fibrosis with crosslink formation were observed at 6 months. On the other hand, the administration of the water intake with LuoHan-Guo fruit extract tended to lower the activity of plasma AST and ALT and decreased the apoptosis of hepatocytes at 2 weeks and significantly reduced the plasma AST and ALT activity at 6 months. From the above results, it has been suggested that Luo-Han-Guo fruit extract can suppress CDAA diet-induced NASH-like lesions in F344 rats. In experiment 2, we developed a mouse NASH model by feeding a modified CDAA (mCDAA) diet,because mice are resistant to the original CDAA diet, and examined effects of Luo-Han-Guo fruit extract dissolved in the drinking water at concentrations of 0, 0.2, 0.6 and 2%. The administration period is set to 7 months. As a result,the control, basal diet group did not show any toxicological changes caused by the administration of Luo-Han-Guo fruit extract. In the mCDAA diet group, NASH-like pathosis, such as steatosis, inflammation and fibrosis, were observed in the liver. Then the co-administration of Luo-Han-Guo fruit extract decreased liver fibrosis and hepatic stellate cell activation in the dose-dependent manner. In experiment 3, we used a dextran sulfate sodium (DSS)-induced colitis models of in mice, in which mucosal damage is induced in the colonic epithelia. DSS was administered in the drinking water for 5 weeks (intermittently every week). Throughout the dosing period of DSS and water, Luo-Han-Guo fruit extract was administered by admixing into a standard chaw at concentrations of 0.15, 0.5 and 1.5%. As a result, the histopathological score of mucosal injury including inflammation, lesion spread and intestinal crypt injury, and decrease in mucous cells, increase in staining of E-cadherin, and increase in number of macrophages were observed as inflammatory changes of mucosal epithelium. On the other hand, mixed administration of Luo-Han-Guo fruit extract showed amelioration or suppression of mucosal damages as mentioned above, and PCNA staining showed increase of the range (cell proliferation zone) consisting of positive cells in the mucosa by the mixed administration of Luo-Han-Guo fruit extract. From the above results, it has been suggested that Luo-Han-Guo fruit extract may have an effect on mucosal regeneration as well as reducing or suppressing the pathological condition of the DSS-induced colitis model in mice. In experiment 4, we evaluated the antioxidant effect of Luo-Han-Guo fruit extract by using the evaluation method of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. As a control, tocopherol and ascorbic acid which are known as antioxidants were used. As a result, although Luo-Han-Guo fruit extract is weaker than tocopherol and ascorbic acid, it shows DPPH radical scavenging activity and was actually confirmed that the antioxidant effect of Luo-Han-Guo fruit extract, and it showed almost the same antioxidant activity as mogroside V. As a result, conferred In summary, Luo-Han-Guo fruit extract has a function of suppressing the occurrence and progression of lifestyle-related diseases. This effect is suggested, at least in part, by virtue of the antioxidant property of its major component, mogroside V.

25－24

Development of food additives stabilizing proteins

Takahiro Muraoka
Tokyo University of Agriculture and Technology

The aim of this study was to develop food additives that stabilize protein to suppress aggregation on the basis of topologically controlled polyethylene glycols. Polyethylene glycol is a linear macromolecule. Topological controls of the physicochemical properties of polyethylene glycol were utilized for this aim. Various polyethylene glycol derivatives bearing different topologies were synthesized. In addition of topologically different polyethylene glycol derivatives, amphiphilic compounds having aromatic units were also synthesized. Detailed studies revealed that the topological differences influenced the properties of polyethylene glycol derivatives. It is also demonstrated that the amphiphilic polyethylene glycols showed efficient protein stabilization effects. This study provides important information for further applications of the polyethylene glycol structure for protein stabilization.

25－25

Controlled release of the flavor components by utilizing amylose conformation and mechanism of the controlled release revealed by solid-state NMR measurement

Keisuke Ueda
Graduate School of Pharmaceutical Sciences, Chiba University

Amylose contained in starch has a spiral structure and can include various small molecules in the space inside of the amylose. In this study, we investigated release inhibiting ability of V-amylose against various flavor components. The sealed-heating of limonene with V-amylose effectively worked for the release suppression of limonene during heating. Solid-state NMR measurements revealed that the molecular mobility of limonene was strongly suppressed in the sealed-heated sample. Moreover, amylose formed molecular interaction with limonene which was included in to the inner cavity of amylose. V-amylose that formed intermolecular interaction with flavor components can be useful for release suppression of flavor components during storage.

25－26

Identification of proteins that interact with volatile compounds in plants and evaluation of their effect to stabilize volatile compounds

Kenji Matsui
Graduate School of Sciences and Technology for Innovation, Yamaguchi University

The purpose of this study is to identify proteins that interact with volatile organic compounds in plants and subsequently to evaluate the effects of the proteins on stabilities of volatile organic compounds. For this purpose, the oil cavities in peels of citrus fruits and the oil bodies in thalli of liverworts were used as the source to seek proteins that potentially interact with volatiles because both the structures are the place where volatile organic compounds are specifically accumulated. With liverworts (Marchantia polymorpha), genome sequence recently published was examined, dozens of terpene synthase-like genes were identified. RT-PCR analysis indicated that there was a subset of terpene synthase-like genes that expressed with high correlation with the formation of terpenoids in oil bodies. By using a fluorescent marker protein, it was confirmed that the terpene synthase-like genes were specifically expressed in oil body cells. One celltranscriptome analysis targeting oil body cells showed that several genes were specifically expressed in oil body cells. One of them, fibrillin-like gene, for example, is likely involved in the storage and/or transport of volatile compounds. With citrus fruit peels, the oil cavities were carefully isolated, and proteins were extracted. SDS-PAGE analysis indicated that the matrix surrounding the cavities contains a 25 kDa-protein. The partial amino acid sequences of the protein were determined, and cloning the corresponding gene is underway. At the same time, analyses on the proteins specific to the content of oil cavities are also underway.

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Analysis and quality evaluation of furanocoumarins contained in essential oil

Masakazu Horie
Department of Food Science, Faculty of Home Economics, Otsuma Women's University

An analytical method using LC-MS/MS of seven furanocoumarins contained in the essential oil was constructed. We investigated the actual condition of commercial essential oils by LC-MS/MS method. As a solvent for dilution,methanol, acetone, tetrahydrofuran (THF) and acetonitrile were investigated. As a result, methanol having the highest solubility, peak shape and peak area was adopted. As a result of analyzing 24 kinds of essential oils marketed in Tokyo, in the case of citrus essential oils, 4 kinds of essential oils including citrus fruits, bergamot, lemon (from Italy,from Argentina), grapefruit (white, pink) And furanocoumarins were detected from 9 kinds of mandarin. In addition,furanocoumarins were also detected in non-citrus lavender (from Tasmania), albeit in a small amount. It was found that there are differences between the same essential oils and varieties, even if they are essential oils collected from the same citrus. Bergapthol and psoralen were not detected in any of the samples. The furanocoumarins bergamottin were detected at relatively higher concentrations than other furanocoumarins.

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Studies on selective adsorption mechanism on gold nanoparticles for removal of odor from long aged sake

Haruno Murayama
Department of Chemistry, School of Science, Kyusyu University

Au nanoparticles supported on various materials such as SiO₂, Al₂O₃, CeO₂, and activated carbon were prepared by the impregnation method using the Au complex coordinated with β-alanine as a precursor. The prepared Au nanoparticles exhibited average diameters less than 5 nm. Au/SiO₂ and Au/Al₂O₃ showed high performance for removal of 1,3-dimethyltrisulfane (DMTS), which is one of the primary components responsible for "hineka". Moreover, Au/SiO₂ selectively reduced the DMTS concentration in long aged sake without decreasing the concentration of sotolone, which is responsible for "jukusei-ko".

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Establishment of Objective Evaluation Method of Swallowing Using Surface Electromyography - Analysis of Correlation between Taste Palatability and Easiness of Deglutition

Tomiko Asakura
University of Tokyo, Department of Applied Biological Chemistry,Graduate School of Agricultural and Life Sciences

Five test solutions derived from the five basic tastes, including preferred and repelled tastes, were subject to sensory evaluation and surface electromyography. Forty-one panelists evaluated the strength, preference, and ease of swallowing these solutions on a nine-step scale. Next, 7.5 mL of each solution was swallowed and surface electromyograms were conducted on the submental muscle at the swallowing point. The model formula was constructed through a PLS regression analysis using each element of surface myoelectric potential as an explanatory variable for preference and ease of swallowing. The formula could predict with high accuracy any taste quality. The importance of the explanatory variable in the model formula differed according to the taste. Furthermore,comparing the ease of swallowing and taste palatability, elements of the surface electromyograms explaining them were different.Bitter and sour tastes tended to coincide with the explanatory elements of palatability and ease of swallowing, but salty, sweet, and umami tastes differed from the explanatory elements of palatability and ease of swallowing. These results suggest that "a favorable taste is easy to swallow, but a bad taste is hard to swallow," which may not always be supported from the biological response perspective.